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Roman, Christopher
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Spring 2018
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2018-03-28
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Doctoral Dissertation
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B cells are central in the development of many autoimmune diseases, such as systemic lupus erythematosus (SLE), through survival and differentiation of autoreactive B cells into antibody-secreting plasma cells. The Christopher Roman lab has previously demonstrated that inhibition of the microphthalmia transcription factor (Mitf), and its family members, Tfe3, Tfeb, and Tfec, in B cells led to splenomegaly (enlarged spleen size), increased splenic plasma cell numbers, and increased serum levels of total IgG and IgM anti-ssDNA autoantibodies. Moreover, inhibition of Mitf and MiT family members in B cells on a murine SLE-susceptible genetic background caused fulminant SLE-like disease characterized by accelerated mortality, production of pathologic autoantibodies, and renal disease. While prior work supported the hypothesis that Mitf and the MiT family restrain B cell activation and prevent emergence of autoreactive B cells, this thesis project characterized two mouse models of Mitf/MiT inactivation and defined mechanisms of gene expression regulation by Mitf that underlie B cell processes of autoreactivity.
Aim 1 characterized two models where: 1) Mitf is not expressed in all cell types and 2) the MiT family is inhibited only in B cells. Studies assessed B cell and T cell subsets (flow cytometry),immunoglobulin and autoantibody serum titers (ELISA), and organization of splenic follicles (areas rich in B lymphocytes; wide-field and confocal microscopy). It was found that MiT inhibition in B cells results in splenomegaly, increased numbers of splenocytes, disorganization of splenic follicles, and increased serum rheumatoid factor. Further, MiT inhibition in B cells increased numbers of pre-B/immature B cells (B220lowCD43low) and plasma cells (CD19-CD38+CD138+) in bone marrow, increased numbers of active helper T cells (CD4+CD62L-CD44high) in spleen, and decreased numbers of naïve helper T cells (CD4+CD63L+CD44low), memory helper T cells (CD4+CCR7+), and marginal zone B cells (CD19+CD93lowCD21highCD23low) in spleen. Mitf inexpression in all cell types, like MiT family inhibition in B cells, led to splenomegaly, increased numbers of splenocytes, disorganization of splenic follicles, increased serum rheumatoid factor, and decreased numbers of memory helper T cells (CD4+CCR7+).Uniquely, Mitf inexpression, but not MiT family inhibition in B cells, resulted in increased germinal center B cells (GL7+CD95+, GL7+CD38+) and increased plasma cells (CD19-CD38+CD138+) in the spleen, as well as decreased numbers ofPD-1+ helper T cells (CD4+CD279+) in spleen, and increased serum levels of Ig Ganti-dsDNA. These results supported the hypothesis that Mitf normally regulates germinal center formation and B cell to plasma cell differentiation.
Aim 2 comprehensively investigated changes of mRNA expression (RNA sequencing) in ex-vivo B cells from both murine models, in which Mitf is not expressed in all cell types, and the MiT family is inhibited only in B cells. It was found that B cells from both models had significantly increased gene expression enriched for the following annotated pathways: regulation of cell cycle (Gene ontology biological process); MHCII antigen presentation (Reactome); and cytokine signaling in the immune system (Reactome). Further, analysis of genes highly overexpressed in B cells in both models reveals that many have been cited for roles in germinal center growth and/or regulation. Other experiments demonstrated that inexpression of Mitf led to increased numbers of B cells with surface expression of activation markers (CD69, CD25)and antigen presentation molecules (MHCII, CD86), and that B cells in culture had increased secretion of TNF-alpha into the supernatant (luminex). RT-qPC Revaluation indicated that Myc expression in-vitro was increased in both models, but that Irf4 and Pou2af1 expression was increased with Mitf inexpression, but not MiT family inhibition.
In conclusion, the presented Thesis demonstrated that Mitf and the MiT transcription factor family normally restrain B cell autoreactivity in murine autoimmune disease through regulation of B cell activation, antigen presentation, cytokine secretion, germinal center organization, plasma cell differentiation, and autoantibody production.
Citation
Amarnani, A. (2018) Mitf and the MiTFamily Restrain B cell Autoreactivity. [Doctoral dissertation, SUNY Downstate Health Sciences University]. SUNY Open Access Repository. https://soar.suny.edu/handle/20.500.12648/15789